Modified bioluminescent proteins and their use

ABSTRACT

A modified bioluminescent protein responds to different physical, chemical, biochemical or biological conditions to produce light or radiation of altered characteristics when the bioluminescent reaction is initiated. The modified bioluminescent protein may respond to modification thereof, e.g. by covalent modification. The protein may include signal peptides to &#34;target&#34; it. DNA coding for the bioluminescent protein may be altered to include tissue specific promoter or enhancer genes so that the altered DNA acts as reporter gene.

This application is a continuation of application Ser. No. 07/820,867,filed Jan. 22, 1992, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to bioluminescent proteins, in particular itrelates to bioluminescent proteins which have been modified, for exampleby chemical means or by genetic engineering. Such modifiedbioluminescent proteins, hereinafter referred to as "rainbow proteins",may be used in the detection and quantification of cells, microbes suchas bacteria, viruses and protozoa, and substances of biological interestsuch as substrates, metabolites, intra- and extra- cellular signals,enzymes, antigens, antibodies and nucleic acids.

Bioluminescence is the oxidation of an organic molecule, the"luciferin", by oxygen or one of its metabolites, to emit light. Thereaction is catalysed by a protein, usually known as a "luciferase", ora "photoprotein" when the luciferin is so tightly or covalently bound tothe luciferase that it does not diffuse off into the surrounding fluid.

    O.sub.2 +luciferin+luciferase→oxyluciferin+light (or O.sub.2  or H.sub.2 O.sub.2) (or photoprotein)

Up to three other substances may also be required to be present in orderto generate light, or to alter its colour, and they are as follows:

(a) A cation such as H⁺, Ca²⁺, Mg²⁺, or a transition metal such as Cu⁺/Cu²⁺, Fe²⁺ /Fe³⁺.

(b) A cofactor such as NADH, FMN, or ATP.

(c) A fluor as an energy transfer acceptor.

Five chemical families of luciferin have been identified so far (seeFIG. 1 of the attached drawing):

(a) Aldehydes (found in bacteria, freshwater limpet Latia andearthworms).

(b) Imidazolopyrazines (found in Sarcomastigophora, Cnidaria,Ctenophora, some Arthropoda, some Mollusca, some Chordata).

(c) Benzothiazole (found in beetles such as fireflies and glowworms).

(d) Linear tetrapyrroles (found in dinoflagellates, euphasiid shrimp,some fish).

(e) Flavins (found in bacteria, fungi, polychaete worms and somemolluscs).

Reactions involving these luciferins may result in the emission ofviolet, blue, blue-green, green, yellow or red light and occasionally UVor IR light and such emission may or may not be linearly or circularlypolarised. Reference is directed to Chemiluminescence principles andapplications in biology and medicine, A. K. Campbell, publ. 1988Horwood/VCH Chichester Weinheim, for further discussion ofbioluminescent reactions.

It has now been found that the light emitted from a bioluminescentreaction involving a modified bioluminescent or "rainbow" protein, maybe changed in intensity, colour or polarisation. Such a change can thenbe used in various assays for detecting, locating and measuring cells,microbes and biological molecules.

In this instance, the cell or substance causes a physical or chemicalchange, such as phosphorylation, to a rainbow protein such as agenetically engineered luciferase, resulting in a change in intensity,colour or polarisation of the light emission. The bioluminescentreaction is triggered by adding, for example, the luciferin, andmodification of the luciferase by the cell or substance being measuredcauses the reaction to emit light at a shorter or longer wavelength.This enables specific reactions to be detected and quantified in livecells, and within organelles or on the inner or outer surface of theplasma membrane, without the need to break them open, and without theneed for separation of bound and unbound fractions.

SUMMARY OF THE INVENTION

According to one aspect of the invention there is provided abioluminescent protein capable of taking part in a bioluminescentreaction to produce light of radiation of altered characteristics underdifferent physical, chemical, biochemical or biological conditions.

The rainbow protein may be produced by the alteration, substitution,addition or removal of one or more amino acids from the end of or withinthe luciferase or photoprotein. As a result the light emission from theoxyluciferin may be of different colours or different states ofpolarisation depending on the physical or chemical conditions. A changein colour to another part of the rainbow spectrum may be induced by:

(a) A change in cation concentration such as H⁺, Ca²⁺, Mg²⁺, ortransition metal.

(b) A change in anion concentration such as Cl⁻ or phosphate.

(c) Covalent modification of the new protein by enzymes causing phospho-or depbosphorylation (including ser/thr, his, and tyr kinases andpbosphatases) transglutamination, proteolysis, ADP ribosylation, gly- orglu-cosylation, halogenation, oxidation, methylation and myristilation.

(d) Binding to the rainbow protein of an antigen, an intracellularsignal such as cyclic AMP, cyclic GMP, Ip3, Ip4, diacyl glycerol, ATP,ADP, AMP, GTP, any oxy or deoxyribonucloside or nucleotide, a substrate,a drug, a nucleic acid, a gene regulator protein.

(e) Expression of its nucleic acid inside a live cell, as well as itsmodification or regulation within the cell by gene expression such aspromoters, enhancers or oncogenes.

Single or multiple mutations and deletions may be detected in a piece ofDNA (eg a PCR product) by linking the "rainbow protein" to one end ofthe DNA and an energy transfer acceptor or quencher to the other end.Nuclease attack at the mutation will separate the rainbow protein fromthe acceptor or quencher and thus cause a change in intensity, colour orpolarisation of the light emission.

Such alteration, substitution, addition or removal of one or more aminoacids may be achieved by chemical means. Alteration of an acid includesalkylation (eg. methylation), phosphorylation and various other covalentmodifications of the type outlined herein. Alternatively the nucleicacid coding for the luciferase or photoprotein may be altered bymodifying, substituting, inserting or deleting one or more nucleotidessuch that the resulting protein has gained or lost a site whichinteracts with the cations, anions, intracellular signal, covalentmodification; proteins or nucleic acid to be measured. The insertion ordeletion of nucleotides is normally produced by site directedmutagenesis or by opening up the gene with a specific restrictionenzyme, inserting or deleting a selected nucleotide sequence and thensealing up of the gene again or using specific primers with thepolymerase chain reaction. The nucleic acid is then transcribed to mRNAand this is then translated to form the rainbow protein either inside abacterial or eukaryotic cell, or in vitro using, for example, rabbitreticulocyte lysate. The new nucleic acid may contain an RNA polymerasepromoter such as T7, SP6, or mammalian promotors such as actin, myosin,myelin proteins, MMT-V, SV40, antibody, G6P dehydrogenase, and can beamplified in vitro the polymerase chain reaction. The result is that therainbow protein can be produced either in a live cell such as a cancercell, or without cells using enzymatic reactions in vitro. The additionof tissue specific promoter or enhancer sequences to the 5' or 3' end ofthe DNA coding for the native or altered bioluminescent protein willenable it to be used as a reporter gene and to be expressed specificallyin a particular cell or tissue, the expression being detectable by theappearance of a change in light intensity, colour or polarisation.

Another way of producing the DNA for a rainbow protein is to separateinto two halves the original DNA for the bioluminescent protein. A pieceof DNA or gene is then inserted between the two halves by ligating onehalf to the 5' end and the other to the 3' end. Alternatively therainbow protein DNA could be generated using the polymerase chainreaction so that the sense primer had one part of the rainbow proteinDNA linked at 5' end and the antisense primer and the other part linkedat the 3' end (i.e. antisense). The pieces of DNA or gene of interest,in the middle, could be from two separate genes. For example one couldcode for am energy transfer protein, the other for a bioluminescentprotein. Only when the two are linked together via a peptide (fromDNA/RNA) will the rainbow protein be generated and a shift in colouroccur. The energy transfer protein could be any fluor bound covalentlyor non-covalently to the protein, for example the green fluorescentprotein from Aequorea, Obelia, Renilla or other cnidarians, or the blueor yellow fluorescent protein from luminous bacteria, or a flavoprotein,or a phyobiloprotein. The whole protein or just the fluorescent demainmay be used. The bioluminescent protein would be any luciferase forexample bacterial, firefly, glowworm or copepod, or any photoprotein forexample aequorin, obelin or a radiolarin such as thalassicollin.

The protein or its DNA or RNA may be incorporated into a live bacteriumor eukaryotic cell by using a virus, plasmid, calcium phosphatetransfection, electropotation, liposome fusion or membrane pore formingproteins. Once inside, only live cells with the appropriate biochemistrywill produce the "rainbow effect". By incorporating the "rainbowprotein" gene into an embryo, oocyte, sperm, seed or seed seeding atransgenic animal or plant may be produced, enabling gene expression,cell regulation, drug action, or cell damage to be located and measuredin individual organs using the "rainbow effect". These new organisms mayalso be used in home aquaria, on aeroplane runways, as safe lights atsea, and as house plants.

The rainbow protein may also be incorporated in a different part of thecell by chemical means or genetically engineering the protein to containa signal peptide which locates it to the inner or outer surface of theplasma membrane or within a particular intracellular organelle (e.g.peroxisome, mitochondrion, chloroplast, endoplasmic reticulum, golgi,secretory vesicle, nucleus or endosome.

Addition of a signal peptide, either chemically or by geneticengineering, will enable the normal or altered luciferase orphotoprotein to be targetted into a specific organelle within the cell,or onto the inner or outer surface of the plasma membrane. For examplethe sequence Met Leu Ser Arg Leu Ser Leu Arg Leu Leu Ser Arg Tyr Leu Leu(SEQ ID No. 1) at the N terminus will locate the bioluminescent proteinin the mitochondria, and Lys Lys Ser Ala Leu Leu Ala Leu Met Tyr Val CysPro Gly Lys Ala Asp Lys Glu (SEQ ID No. 2) on the N terminus will targetthe protein to the endoplasmic reticulum, a Lys Asp Glu Leu (SEQ ID No.3) sequence at the C terminus retaining it there.

The initial luciferase or photoprotein or its gene may come from any ofthe known chemistries in bioluminescence (see FIG. 1) or from the widerange of uncharacterised luminous organisms from more than 700 generarepresenting at least 16 phyla. The luciferin may be synthesisedchemically and added to the biological reaction or cell to generatelight. Aternatively, the gene coding for the enzymes responsible formaking the luciferin may be linked to the "rainbow protein" gene so thatthe artificial operon or fusion gene expresses both rainbow protein andmakes luciferin in the live cell from normal cell constituents such asamino acids.

According to a second aspect of the invention there is provided a methodof producing a bioluminescent protein by altering, substituting, addingor deleting one or more amino acids to the protein by chemical means orby genetically engineering the nucleic acid coding for the protein.

According to a further aspect of the invention there is provided nucleicacid coding for the bioluminescent protein as hereinbefore defined.

The rainbow protein, or the nucleic acid coding for it, may be used in arange of biological investigations; such as:

(a) Detection, location and measurement of microbes (protozoa, bacteria,viruses).

(b) Detection and location of cancer cells.

(c) Measurement of enzymes, intracellular signalling and other turnoverreactions in cells or fluids.

(d) DNA and RNA binding assays.

(e) Immunoassay and other protein binding assays.

The rainbow proteins and their parent nucleic acids also may be used ingenetic engineering, in the development of transgenic animals, plantsand microbes, and in horticulture.

According to yet a further aspect of the invention there is provided theuse of a rainbow protein, or the nucleic acid coding for the rainbowprotein, for the detection, location or measurement of substances ofbiological interest such as microbes, cells or biological molecules orreactions therein.

In this aspect, the reaction or substances of biological interest aremade to interact with the rainbow protein or its parent nucleic acid.Such interactions include direct or indirect linking such asnon-covalent or covalent binding as well as energy transfer processes.

Although the invention has been described above it is to be understoodthat it includes any inventive combination of the features set out aboveor in the following description.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 indicates five chemical families of Luciferin.

The invention may be performed in various ways and will be furtherdescribed with reference to the following examples:

EXAMPLE 1 Detection of Phosphorylation of a Rainbow Protein

The peptide leu arg arg ala ser leu (SEQ ID No. 4), known as kemptide,or Arg Thr Lys Arg Ser Gly Ser Val Tyr Glu Pro Leu Lys Thr (SEQ ID No.5) known as malantide was covalently linked to firefly luciferase usingdisuccinyl suberate at pH8. Addition of 125 ul protein kinase A+cyclicAMP (200 uM)+125 uM ATP caused phosphorylation of the kemptide, nowattached to the luciferase. The resulting shift in colour fromyellow-green to red at pH 7.8 or from red to yellow-green at pH 6.5,measured as a ratio in a dual wavelength chemiluminometer enabled therate of protein phosphorylation by the kinase to be assayed, anddephosphorylation induced by phosphatase to be assayed subsequently bythe reversal of the ratio.

EXAMPLE 2 Engineering Firefly Luciferase

cDNA coding for firefly luciferase was amplified using the polymerasechain reaction (PCR) using 5' sense primers with a T7 RNA polymerasepromoter, and 3' antisense primers as follows: Primer code in brackets

5' Sense Primers

(105) CACCTAATACGACTCACTATAGGGAGAATGGAAGACGCCAAAAAC (SEQ ID NO. 6)

(107) AGAACTGCCTGCCGCAGATTCTCGCA (SEQ ID NO. 7)

(110) ATGCTGTCCCGGCTGTCCCTGCGGCTGCTGTCCCGGTACCTGCTGAAGACGC CAAAAAC (SEQID NO. 8)

(111) CACCTAATACGACTCACTATAGGGAGAATGCTGTCCCGGCTGTCC (SEQ ID NO. 9)

3' Antisense Primers

(100) TCTCGCTGAATACAGTTAC (SEQ ID NO. 10)

(106) CCCCAAGCTTAGATCTCTCTGATTTTTCTTGCGT (SEQ ID NO. 11)

(108) TGCGAGAATCTGCGGCAGGCAGTTCT (SEQ ID NO. 12)

The following firefly luciferase cDNA's were constructed using primersin brackets:

(a) full length (105+100)

(b) -36 bp i.e. missing peroxisomal signal peptide (105+106)

(c) protein kinase A site (Arg Arg Xaa Sec) (SEQ ID NO. 13) in middle ofprotein (step 1: 105+108 and 107+100; step 2:2 halves from step1+105+100)

(d) mitochondrial signal at N terminus (step 1:110+100; step 2: step 1sample+111+100).

The PCR reaction in 50 μl contained 10 mM Tris pH 8.3, 0.01% gelatin,0.4U Taq polymerase, 2 mM MgCl₂, 0.2 mM each dATP, dGTP, dTTP, dCTP, 0.5μM of each primer, 1 μl DNA (ca 1-100 ng). The reaction, covered with 50μl mineral oil, was incubated in a Perkin-Elmer thermal cycler for 25cycles: 94° C. 1 minute, 55° C. 1 minute, 72° C. 2 minutes+5 secondsextension on each cycle, then for 30 minutes at 37° C. with 1U E.coliDNA polymerase (Klenow fragment).

Successful PCR was confirmed by a band on agarose gel electrophoresis.The cDNA was purified by centricon to remove primers, and precipitatedin 70% ethanol, 0.7 mM NH₄ acetate after extraction with bufferedphenol: CHCl₃ : secondary amyl alcohol (25:24:1). The DNA (0.5-1 μgdissolved in 10mM Tris 0.1 or 1 mM EDTA pH 7.4-7.5) was transcribed inthe T7 RNA polymerase in buffer containing 40 mM Tris, pH 7.4-7.5, 6mMMgCl₂, 10 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 2 mMspermidine, 0.5 mM each ATP, CTP, UTP, 0.1 mM GTP, 0.5 mM cap m7 G(5')ppp (5') G, 1000 U RNAsin/ml, 800 U T7 RNA polymerase±2 μC, ^(32P) UTPfor up to 4 hours (1-2 hours optimal), at 37° C. The reaction wasstopped in the ice cold phenol: CHCl₃ : secondary amyl alcohol(25:24:1), and the RNA precipitated in 70% ethanol+0.7M NH₄ Ac, andstored at -20° C.

The RNA was centrifuged, redissolved in 20 μl 70 mM Tris, 1 mM EDTA pH7.4-7.5 and 1 μl incubated with 5-10 μl rabbit reticulocyte lysate for 1hour at 30° C. to synthesize the luciferase. Luciferase, after dilution,1/100 is assayed for light emission directly in 50 mM tris, 10 mM MgAc₂,0.1 mg/ml bovine serum albumin, 0.1-0.2 mM luciferin, 0.5-5 mM ATP, pH7.8, or isolated by isoelectric focusing. The mutant (Arg Arg Xaa Sec)(SEQ ID NO. 13) luciferase has a pI of ca 7.1 or 6.8, and the normalluciferase pI ca 6.8. The luciferase with mitochondrial signal alsoseparated from the normal luciferase. On addition of the rabbitreticulocyte lysate containing this altered luciferase it was taken upby added mitochondria, as shown by centrifugation and light emissionfrom the mitochondria and luciferase.

Phosphorylation of the Arg Arg Xaa Sec (SEQ ID NO. 13) containingluciferase with protein kinase A, cyclic AMP (0.2 mM), ATP (0.1-1 mM)pH7, caused the luciferase to change its pI back towards 6.8, and toshift its colour. The RRXS luciferase had a greener light emission thanthe native luciferase detected using a dual wavelength chemiluminometerwith interference filters (maximum transmission ca 545 and 603 mM).

Primers containing kemptide nucleotide sense or antisense sequence (LeuArg Arg Ala Ser Leu Gly) (SEQ ID No. 4); or malantide Arg Thr Lys ArgSer Gly Ser Val Tyr Glu Pro Leu Lys Ile (SEQ ID No. 14) were also addedeither to N or C terminus using a one or two step PCR reaction. Thesealso produced luciferase which could be phosphorylated thereby alteringits intensity and colour.

EXAMPLE 3 Preparation of Engineered Aequorin

cDNA or genomic coding for the Ca²⁺ -activated photoprotein was PCR'd ina similar way to that for firefly luciferase. Using one or two step PCRthe protein kinase A recognition peptide kemptide (LRRLALG) or malantide(as Example 2) was added to the N terminus. The mutant aequorin haddifferent kinetic properties enabling protein kinase A to be detected byphosphorylating the altered aequorin (above).

Normal aequorin primers =5' sense TAATACGACTCACTATAGGGGAGAGAATGGTCAAGCTTTACATCAGACTTCGAC, (SEQ ID NO. 15) and 3'antisense GAATTCTTAGGGGACAGCTCCACCGTA. For insertion (SEQ ID NO. 16) ofkemptide at the N terminus the nucleotide sequence equivalent to Leu ArgArg Ala Ser Leu Gly (SEQ ID No. 4) was attached to the first 15 bases(including ATG) of the 5' end of aequorin. In step 2, the T7 RNApolymerase promoter was added to form the kemptide-aequorin in vitro forin vitro phosphorylation. Genomic aequorin DNA (made by PCR) was atleast as active as that made from mRNA by reverse transcriptase PCR.

EXAMPLE 4 Detection of Cancer Cells in Blood

A blood sample (1 ml) is mixed with, a suspension of liposomescontaining mRNA coding for a rainbow protein catalysing thebenzothiazole reaction c in FIG. 1. This mRNA was produced as follows:

The gene coding for firefly luciferase was first isolated from a cDNAlibrary in E. coli using pCDV1 plasmid primer+Honjo linker containingSP6 RNA polymerase promoter. A nucleotide sequenceGCTCGTCTTATTGAAGATAATGAATATACTGCTCGTTTTGGT representing thephosphorylation site for tyrosine kinase activity of the myc oncogenewas inserted at the EcoRI restriction site 30 base pairs downstream fromthe ATG at the 5' end. The DNA was resealed, recloned and the plasmidinsert transcribed by SP6 RNA polymerase in vitro to produce mRNA forthe rainbow protein. The original protein produced yellow-green lightbut the rainbow protein when phosphorylated in the cell produces redlight. Thus the presence of cancer cells was detected in blood samplefrom a leukaemia patient by measuring the ratio of yellow-green (545 nm)to red (603 nm) light in a dual wavelength chemiluminometer.

EXAMPLE 5 Detection of Salmonella

The cDNA for the rainbow protein in Example 4 containing SP6 RNApolymerase promoter was inserted into Salmonella phage. Addition of thisphage to Salmonella resulted in expression of the rainbow protein andthe generation of red light, enabling as few as 1 bacterium per 20 ml tobe detected.

EXAMPLE 6 Detection of HIV RNA

A sample (1 ml) of blood from a patient with AIDS was extracted with 4Mguanidium isothiocyanate and the nucleic acid precipitated withethanol/NH acetate. An oligonucleotide (10 ul, 1 uM) labelled with arainbow protein generated from the photoprotein obelin was added to 100ul redissolved RNA at 50° C. and the mixture cooled for 10 minutes at 0°C. The oligonucleotide was specific for a sequence in the HIV coatprotein. Binding this to HIV RNA resulted in a shift in the lightemission from the rainbow protein from light blue (475 nm) to blue (440nm). This was detected as a shift in the ratio of light emission atthese two wavelengths in a dual photomultiplier chemiluminometer.

EXAMPLE 7 Measurement of Testosterone in Blood

Testosterone carboxyoxime is reacted with the rainbow protein from thephotoprotein obelin to form a testosterone rainbow protein conjugate. 5ul of this containing 1 nmol was incubated with a solution of antibodylabelled with fluorescein to testosterone (50 ul) pH 7.4 for 30 minutesin the presence or absence of varying concentrations of standardtestosterone. The bioluminescent reaction was triggered by addition ofCa and the ratio of light at 475 nm to 530 nm measured. Increasing theconcentration of standard testosterone increased the ratio at 475/530.This procedure could be carried out without the need to separate boundfrom free antigen.

EXAMPLE 8 Detection of Listeria

A sample of suspect food is boiled to extract DNA. A sense primer to thespecific Listeria gene or domain covalently coupled to obelin cDNA+5P6RNA polymerase promotor and antisense primer covalently coupled toantisense green fluorescent protein (GFP) cDNA is used to amplify theListeria gene using the polymerase chain reaction. The result is DNAcoding for a new rainbow protein and transcribable by SP6 RNApolymerase. ##STR1## This DNA is transcribed and the mRNA translatedusing rabbit reticulocyte lysate. Coelenterazine is added to reactivateobelin. The ratio of light at 510/475 nm for rainbow protein versusobelin alone is directly proportional to the amount of Listeria DNAoriginally present in the food sample. When no Listeria are present theratio of ratios is 1.

EXAMPLE 9 Measurement of Nucleic Acid Hybridisation by Polarisation

The reaction described in Example 6 was carried out but the lightemission was detected in a dual photomultiplier chemiluminometercontaining two plane polarised filters with the polarisation planes at90° to each other. The ratio between the two photomultipliers wasrelated to the amount of HIV RNA present.

EXAMPLE 10 Measurement of Cyclic AMP or Ip3

Using a two step PCR reaction as described in Examples 2 and 3, thecyclic AMP binding domain from the bacterial CAP protein or the Ip3binding domain of the endoplasmic reticulum receptor was added to the Nor C terminus or into firefly luciferase or aequorin. The alteredproteins were made in vitro from the PCP DNA product as described inexamples 2 and 3 and characterised by activity and colour of lightemission cyclic AMP or Ip3. A change in both intensity and colourenabled CAMP or Ip3 to be measured in cell extracts out in living cells.Using an image intensifier a CAMP or Ip3 "cloud" could be visualised inthis one cell, Similarly the aequorin or luciferase could be seen withinthe ER or a mitochondrion if it was first made with an EP ormitochondrial signal attached to it (±KDEL) at the C terminus.

It will be appreciated that the bioluminescent protein may besynthesised from amino acid sequences or using DNA or RNA synthesistechniques, instead of by modification of a protein produced by anorganism.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 17                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetLeuSerArgLeuSerLeuArgLeuLeuSerArgTyrLeuLeu                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       LysLysSerAlaLeuLeuAlaLeuMetTyrValCysProGlyLysAla                              151015                                                                        AspLysGlu                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       LysAspGluLeu                                                                  (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       LeuArgArgAlaSerLeuGly                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ArgThrLysArgSerGlySerValTyrGluProLeuLysThr                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CACCTAATACGACTCACTATAGGGAGAATGGAAGACGCCAAAAAC45                               (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AGAACTGCCTGCCGCAGATTCTCGCA26                                                  (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ATGCTGTCCCGGCTGTCCCTGCGGCTGCTGTCCCGGTACCTGCTGAAGACGCCAAAAAC59                 (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CACCTAATACGACTCACTATAGGGAGAATGCTGTCCCGGCTGTCC45                               (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TCTCGCTGAATACAGTTAC19                                                         (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CCCCAAGCTTAGATCTCTCTGATTTTTCTTGCGT34                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TGCGAGAATCTGCGGCAGGCAGTTCT26                                                  (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ArgArgXaaSer                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ArgThrLysArgSerGlySerValTyrGluProLeuLysIle                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      TAATACGACTCACTATAGGGGAGAGAATGGTCAAGCTTTACATCAGACTTCGAC54                      (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GAATTCTTAGGGGACAGCTCCACCGTA27                                                 (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GCTCGTCTTATTGAAGATAATGAATATACTGCTCGTTTTGGT42                                  __________________________________________________________________________

What is claimed is:
 1. A modified bioluminescent protein selected fromthe group consisting of firefly luciferase, aequorin and obelin,modified to include an interaction site for a selected analyte, saidsite being not present in the unmodified protein, wherein the analyteinteracts with said interaction site, the bioluminescent protein beingreactive in a bioluminescent reaction to produce light, wherein thelight is produced having a physical property of a first characteristicwhen the analyte is interacting with the interaction site, and isproduced having a second different characteristic of said physicalproperty when the analyte is not interacting with the interaction site;said interaction site being selected from the group consisting ofkemptide covalently bound to the N or C terminus of firefly luciferase,kemptide covalently bound to the N terminus of aequorin, kemptidecovalently bound to the N terminus of obelin, malantide covalently boundto the N terminus of aequorin, and an RRXS phosphorylation site at aminoacid 217 of firefly luciferase.
 2. A bioluminescent protein according toclaim 1 for intracellular use, wherein said protein includes a signalpeptide for targeting said protein to a specific organelle or sitewithin a cell.
 3. A modified bioluminescent protein selected from thegroup consisting of firefly luciferase, aequorin and obelin, modified toinclude an interaction site for a substance of interest, said site beingnot present in the unmodified protein and reactive in a bioluminescentreaction, wherein light emitted in said bioluminescent reactioninvolving said protein changes depending on the concentration of saidsubstance of interest; said interaction site being selected from thegroup consisting of kemptide covalently bound to the N or C terminus offirefly luciferase, kemptide covalently bound to the N terminus ofaequorin, kemptide covalently bound to the N terminus of obelin,malantide covalently bound to the N terminus of aequorin, and an RRXSphosphorylation site at amino acid 217 of firefly luciferase.
 4. Nucleicacid coding for the bioluminescent protein as claimed in claim
 1. 5.Nucleic acid according to claim 4, including promoter or enhancersequences specific to a tissue or substance of interest.
 6. Nucleic acidaccording to claim 4, including a nucleotide sequence coding for anenzyme for producing luciferin.
 7. A method of detecting a change in aphysical, chemical, biochemical or biological condition, the methodcomprising:providing a bioluminescent protein according to claim 1 in areaction system containing an analyte of interest; observing whetherlight of the first and/or second characteristic is produced; anddetecting said change based on production of light of said first and/orsecond characteristic.
 8. A method of detecting, locating or measuring asubstance of biological interest, the method comprising:(a) providing abioluminescent protein according to claim 1 in a reaction systemcontaining an analyte of interest; (b) analyzing said first and/orsecond characteristics of the light produced; and (c) detecting,locating or measuring said analyte based on said first and/or secondcharacteristic of the light produced.
 9. A method of detecting,locating, measuring a substance of biological interest within a cellwhich comprises:introducing into said cell nucleic acid coding for abioluminescent protein according to claim 1, causing said modifiedbioluminescent protein to be expressed, causing said bioluminescentprotein to take part in a bioluminescent reaction, analyzing said firstand/or second characteristics of the light produced; and detecting,locating or measuring said analyte based on said first and/or secondcharacteristic of the light produced.
 10. A method according to claim 9,wherein said nucleic acid includes a promoter or enhancer sequencespecific to a substance of interest.